We are studying the reactions required for DNA replication in vitro using proteins encoded by T4 bacteriophage. With single-stranded phy x 174 DNA as a model template, we find that a heterogeneous family of pentaribonucleotides with the sequence pppACNNN are made by the T4 gene 41 protein and a second protein controlled by gene 61. These primers are elongated to form duplex circular molecules by the gene 43 DNA polymerase, gene 45 protein, and the complex of gene 44 and 62 proteins (a DNA-dependent ATPase). If the gene 32 DNA binding protein is also present, the DNA is further elongated by strand displacement synthesis yielding rolling circles with long tails. The priming activity associated with gene 61 copurifies with a basic protein which has a native and natured molecular weight of about 42,000. We are searching in vivo and in vitro for T4 proteins required for the initiation of DNA replication within duplex templates. We have identified and partially purified a nuclease controlled by a late T4 protein which hydrolyzes T7 and lambda DNA to a series of discrete sized products.